Process for the production of acid protease by cultivation of microorganisms



Jan. 27, 1970 yos n-ns KOAZE ETAL 3,492,204

PROCESS FOR THE PRODUCTION OF ACID PROTEASE BY CULTIVATION OF MICROORGANISMS Filed March 30, 196 6 8 Sheets-Sheet l Ffjl ' INVENTORS Yaw/H054 5 0425 1647-417 Gal 227K566? flmea Jan. 27, 1970 YQ$H|H|sA KQAZ ETAL 3,492,204

PROCESS FOR THE PRODUCTION OF ACID PROTEASE BY CULTIVATION OF MICROORGANISMS Filed March 30, 1966 8 Sheets-Sheet 2 Fig- OPTIC/ll. DENSITY Q Q Ln 0 l l l l WAVELENGTH W) (Moo Wan Kym/01a 1% Arm 26B.

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PROCESS FOR THE PRODUCTION OF ACID PROTEASE BY CULTIVATION OF MICROORGANISMS Filed March 30, 1966 8 Sheets-Sheet 5 50 8F zm wc) INVENTOR: yOJH/H/Jfl A6425, A A ww/ 60/ Jan. 27, 1970 YQSHIHISA KOAZE ETAL 3,492,204

PROCESS FOR THE PRODUCTION OF ACID PROTEASE BY CULTIVATION OF MICROORGANISMS Filed March 30, 1966 8 Sheets-Sheet 4 ELY-55 E66! ALBUM/N F29 -54 M/LK CASE/N ALID-DENATURED HEMOGLOB/N (/REA-DENHTURED HEMOGLOB/N 1 N VEN TORS )(asH/H/JA 16426, Afraavw 60/ BY EKG'SH/ 14 4124 Wasoufiwnwfi; Swami A 7' T0? N5 V5 Jan. 27, 1970 YQSHIHISA Ko z ETAL 3,492,204

PROCESS FOR THE PRODUCTION OF ACID PROTEASE BY CULTIVATION 0F MICROORGANISMS 8 Sheets-Sheet 5 BLOOD F/BR/N Filed March 30 1966 WHE/l T GLUTEN 27, 1 0 YOYSHIHISA KOAZE ETAL 3,492,204

PROCESS FOR THE PRODUCTION OF ACID PROTEASE BY CULTIVATION OF MICROORGANISMS Filed March 50 1966 8 Sheets-Sheet 6 BY 7Zw6ow/ Al ie/9 Jan. 27, 1970 vos u-us KOAZE ETAL 3,492,204

PROCESS FOR THE PRODUCTION OF ACID PROTEASE BY CULTIVATION 0F MICROORGANISMS Filed March 30 1966 8 Sheets-Sheet 7 1 ACID PHONE/95E A m IIMPERATl/RE (c) HC/D P20720515 B 7/14 w 6} 72M 072 Q masm/ mules) d mmmm 4 "Fro/2N5 Vs YOSHIHISA KOAZE ErAL 3,492,204 PROCESS FOR THE PRODUCTION OF ACID PROTEASE Jan. 27, 1970 BY CULTIVATION OF MICROORGANISMS Filed March 50, 1966 8 Sheets-Sheet 8 w IO 2 -w H V 0 B 7 0 MM -6 E -w 0 -0 W 4 w 0 C 5 A 0 M Wm 2 -0 1 V .9 m 00 0 my, 7 m -w w -m P 0 w 4 C TENPERA TURE m TENPERATURE (C mama n--:+- -----u/iz/L 5mm 1 N VEN TORS' J/IIH/JA 15425 647057 62/ g Y 727K656? #[4/24 mam awswoa United States Patent 3,492,204 PROCESS FOR THE PRODUCTION OF ACID PROTEASE BY CULTIVATION OF MICROORGANISMS Yoshihisa Koaze, Tokyo, Hitoshi Goi, Kawasaki-ski, and Takeshi Hara, Tokyo, Japan, assignors to Meiji Serka Kaisha, Ltd., Tokyo, Japan, a corporation of Japan Filed Mar. 30, 1966, Ser. No. 538,715 Claims priority, application Japan, Mar. 31, 1965, 40/ 18,279, 40/ 18,280 Int. Cl. C12k 1/10; C12d 13/10; C07g 7/02 US. Cl. 19566 4 Claims ABSTRACT OF THE DISCLOSURE The production of acid protease A and acid protease B is accomplished by cultivating a strain of Aspergillus niger in a culture medium containing a greater concentration of organic nitrogen than used formerly. Such cultivation produces acid protease A having an optimum active pH at pH 2.0, 30 C. and a high activity at pH 1.5, 55 C. and acid protease B having an optimum active pH at pH 2.6, 30 C. and substantially no activity at pH 1.5, 55 C.

This invention relates to a process for the production of acid protease by cultivation of microorganisms. In particular, the present invention relates to the process for the production of acid protease B which has an optimal active pH at pH 2.6 and loses its activity at pH 1.5 and at a temperature above 50 C. and new high acid protease A which has an optimal active pH at pH 2.0 and exhibits high activity even at pH 1.5 and at a temperature above 50 C. by both solid and liquid cultivation of a strain belonging to family Aspergillaceae including mainly Aspergillus niger group, using a culture medium with considerably increased amount of organic nitrogen source.

Throughout the following specification each reference to the strain DBD0406, mentioned above, is to be read as DBD-0406 (ATCC 16513).

FIGURE 1 is a reproduced diagram of electron microscopic photograph of the conida of the strain DBD0406 belonging to Aspergillus niger var. macrosporus.

FIGURE 2 is a reproduced diagram of optical micro scopic photograph of the conidia of the strain DBD-0406 belonging to Aspergillus niger var. macrosporus.

FIGURE 3 is an ultraviolet absorption spectrum of new high acid protease A and acid protease B.

FIGURE 4 is the curves showing phenomena of temperature and pH on proteolytic activity of the new acid protease.

FIGURES 5A, B, C, D, E, F, G and H show the effects of pH on various protein digestions by acid protease A and B indicated by lines A and B respectively.

FIGURE 6 is a pattern of amino acids analysis on milk casein hydrolyzed with acid protease B.

FIGURE 7 is a pattern of amino acids analysis on milk casein hydrolyzed with acid protease A.

FIGURES 8A, B show the effects of temperatures on milk casein digestion by acid proteases A and B.

FIGURES 9A, A, B, B show the head and pH stabilities of acid proteases A and B.

We have found from our studies on the enzymes produced by microorganisms that a new strain Aspergillus niger var. macrosporus DBD-0406 belonging to Asperice gillus niger group isolated from soil by using a special acid medium may produce both acid protease B and new high acid protease A at the same time,.acid protease B having an optimal active pH at pH 2.6 and losing its activity at pH 1.5 and at a temperature above 50 C. and new high acid protease A having an optimal pH at pH 2.0 and exhibiting high activity even at pH 1.5 and at a temperature above 50 C., and have succeeded to make the production of said both acid protease rapid progress by considerably increasing the amount of organic nitrogen source in both solid medium and liquid medium.

The microbial characteristics of Asperg illus niger var. macrosporus DBD-0406 strain is as follows:

(4) Diameter of conidia 4.9-5.9 (average 5.4g).

(5) Vesicles Globose, rarely subglobose.

(6) Diameter of vesicles 58-70,u (average 67a).

(7) Length of 1st sterigmata 18-30,:1. (average 26 (8) Length of 2nd sterigmata 5-8 (average 7a).

(9) Length of conidiophores". 1.75-3.13 mm. (average The above strain was deposited in the American Type Culture Collection on Mar. 30, 1966, under the number of 16513.

The shape of the conidia of Aspergillus niger var. macrosporus is shown in FIGURES l and 2.

According to the present invention we have succeeded to make the production of acid protease A twice or more usual production in the solid cultivation as shown in Tables 1-3 by making organic nitrogen content in the medium containing organic nitrogen sources only above 2.5% by adding organic substance having comparatively low nitrogen content such as bran together with the organic substance having comparatively high nitrogen content such as defatted soy bean meal or making organic nitrogen/inorganic nitrogen in the medium containing both organic and inorganic nitrogen sources above 4 by combined use of inorganic nitrogen source and organic nitrogen source, the inorganic nitrogen source such as ammonium sulfate and the like being made 0.6% as inorganic nitrogen and the organic nitrogen source being composed of the organic substance having comparatively low nitrogen content such as bran and the organic substance having comparatively high nitrogen content such as defatted soy bean meal which are mixed in such an amount that the organic nitrogen content may become above 2.5

Components of medium (g.)

lABLE 2.EFFECT OF INCREASE OF ORGANIC NITROGEN IN COMBINATION OF OR- GANIC AND INORGANIC NITROGEN IN A CULTURE MEDIUM ON THE PRODUC- TION OF ACID PRoTEhisE A BY THE CULTIVATION OF ASPERGILLUS NIGER VAR.

MA C'ROSPOR US DlBD-04 Components of medium Organic Organic Inorganic nitrogen] Productivity Deiatted soy- Ammonium nitrogen nitrogen inorganic of acid Bran (g.) bean meal (g.) sulfate (mg.) (percent) (percent) nitrogen protease A TABLE 3.-EFFECT INCREASE OF ORGANIC NITROGEN IN COMBINATION OF OR- GANIC AND INORGANIC NITROGEN IN A CULTURE MEDIUM OM THE PRODUC- TION OF ACID PROTEASE A BY THE CULTIVATION OF ASPERGILLUS NIGER VAR.

MACR OSP ORUS DBD-0406 Components of medium Organic 7 Organic Inorganic nitrogen] Productivity Defatted soy- Ammonium nitrogen nitrogen Inorganic of acid Bran (g.) bean meal (g.) sulfate (mg) (percent) (percent) nitrogen protease A Further in this case the yield of acid protease may 1e increased more than --60% or up to near twice as .hown in Table 4 by adding water to the mediumin an tIIlOUnt more than equal to the content of the solid matter of the medium.

FABLE 4.EFFECT OF THE AMOUNT OF WATER ADDED TO SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROIEASE A BY THE CULTIVATION OF ASPER- GILLUS NIGER VAR. MACROSPOR US DBD-0406 added water 80% 100% 120% Ratio of the production of acid protease A:

Culture medium 1 100 133 222 Culture medium 2 100 124 153 Culture medium 1: Mixture of bran and defatted soybean meal (1:1). Culture medium 2: Mixture of bran and defatted soybean meal (7:3).

yer mut. shiema'nni, Aspergillus 'riureusvar. acidus. Asi uergillu's aureus var. 7 br'vis} Aspergillus bdraiae, Pencillium frequentans and Pencillium thomii have been ob-' served to show excellent results as shown in Table 5. p

CULTURE MEDIUM ON TABLE 5.-EFFECT OF INCREASE OF ORGANIC NITRO- GEN SOURCES IN A SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE A BY THE CULTIVA- TION OF SEVERAL STRAINS BELONGING TO FAMILY ASPERGILLACEAE Productivity of Acid protease A,

Culture medium 1: Bran only, added water (organic nitrogeni cirl ture medium 2: Mixture of bran and deiatted soybean meal (1:1) added water 120% (organic nitrogen: 5%).

Further we have succeeded to make the production of acid protease A twice or more than usual production in the liquid cultivation too as shown in Table 6 by making the nitrogen content in the medium containing organic nitrogen sources only above 0.4% or in case the medium contains both organic nitrogen source and inorganic nitrogen source, making the organic nitrogen/inorganic nitrogen in the :medium more than: 6 by using the inorganic nitrogen source such as ammonium sulfate in such an amount as the nitrogen content thereofcome to below 0.06% and by adding the organic nitrogen source such as defatted soy bean meal, corn steep liquor in such considerable amount as the nitrogen content thereof come to more than 0.4% Y

TABLE senrrnc'r or INo r EAsE OF ORGANIC NITROGEN SOURCES IN A LIQUID E PRODUCTION OF ACID PROTEASE A BY THE CUL- TIVATION OF ASPERGILLUS NIGER VAR. MAOROSPORUS DBD-0406 Nitrogen sources (percent) 7 Organic Organic Inorganic nitrogen/ Productivity Defatted I Corn steep Ammonium nitrogen nitrogen Inorganic of acid soybean meal liquor sulfate (percent) (percent) nitrogen, protease A According to our studies on the production of acid protease A of the known fungi by applying this new liquid culture method, aforesaid Aspergillus niger group except Penicillium frequentans and Pen. thomii have been observed to show excellent results as shown in Table 7.

TABLE 7.-EFFECT OF INCREASE OF ORGANIC NITROGEN SOURCES IN A LIQUID CULTURE MEDIUM ON THE PRO- DUCTION OF ACID PROTEASE A BY THE CULTIVATION gggEVERAL STRAINS BELONGING TO BLACK ASPER- Productivity of acid protease A, percent, culture media Culture medium 1: Starch 1%, defatted soy bean meal 3%, corn steep liquor 1%, K2HOP4 0.1%, 02100 1%.

Culture medium 2. Detatted soy bean meal 6% instead of 3% 1n the culture medium 1.

Culture medium 3: Defatted soy bean meal 8% instead of 3% in the culture medium 1.

Culture medium 4: Ammonium sulfate 0.3% is added to the culture medium On the other hand, according to the present invention We have succeeded to increase the production of acid protease B in the solid cultivation more than 40-60% or up to near twice as shown in Tables 8-14 by making the organic nitrogen content in the medium containing organic nitrogen sources only above 4% by combined use of the organic substance having comparatively low nitrogen content such as bran and the organic substance having comparatively high nitrogen content such as defatted soy bean meal, corn steep liquor, defatted cotton seed meal or making organic nitrogen/ inorganic nitrogen in the medium containing both organic and inorganic nitrogen sources above 6 by combined use of inorganic nitrogen source and organic nitrogen source, the inorganic nitrogen source such as ammonium sulfate being made below 0.8% as inorganic nitrogen and the organic nitrogen being composed of the organic substance having comparatively low nitrogen content such as bran and the organic substance having comparatively high nitrogen content such as defatted soy bean meal, defatted cotton seed meal which are mixed in such an amount that the organic nitrogen content may become above 4%.

TABLE 8.EFFECT OF INCREASE OF ORGANIC NITROGEN IN A SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY THE CULTIVATION OF ASPER- GILLUS NIGER VAR. MACROSPORUS Deiatted Organic cotton seed nitrogen Productivity of Bran (g-) meal (g.) (percent) acid protease B TABLE 9.EFFECT OF INCREASE OF ORGANIC NITROGEN IN A SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY THE CULTIVATION OF ASPER- GILLUS NIGER var. MACROSPORUS BED-0406 Deiatted Organic Productivity soy bean nitrogen of acid Bran (g.) meal (g.) (percent) protease B TABLE 10.EFFECT OF INCREASE OF ORGANIC NITRO- GEN IN A SOLID CULTURE MEDIUM ON THE PRODUC- TION OF ACID PROTEASE B BY THE CULTIVATION OF ASPERGILLUS NIGER VAR MACROSPORUS DEB-0406 TABLE 11.-EFFECT OF INCREASE OF ORGANIC NITROGEN IN COMBINATION OF OR- GANIC NITROGEN AND INORGANIC NITROGEN IN A SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY THE CULTIVATION OF ASPERGILLUS NIGER VAR. MA CROSPOR US DEB-0406 Composition of medium Inorganic Organic nitro- Productivity Deiatted soy- Ammonium Organic nitronitrogen (pergen/inorganic of acid Bran (g.) bean meal (g.) sulfate (mg) gen (percent) cent) nitrogen protease B TABLE 12.EFFECT OF INCREASE OF ORGANIC NITROGEN IN COMBINATION OF OR- GANIC NITROGEN AND INORGANIC NITROGEN IN A SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY THE CULTIVATION OF ASPERGILLUS NIGER VAR. JMACROSPORUS DBD-0406 Composition of medium Inorganic Organic nitro- Productivity Defatted soy- Ammonium Organic m'tronitrogen (pergen/inorganic of acid Bran (g.) bean meal (g.) sulfate (mg.) gen (percent) cent) nitrogen protease B TABLE 13.EFFECT OF INCREASE OF ORGANIC NITROGEN IN COMBINATION OF OR- GANIC NITROGEN AND INORGANIC NITROGEN IN A SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY THE CULTIVATION OF ASPERGILLUS NIGER VAR. .MAC'ROSPORUS DBD-0406 Composition of medium Inorganic Organic nitro- Productivity Defatted soy- Ammonium Organic nitronitrogen (pergen/inorganic of acid Bran (g.) bean meal (g.) sulfate (mg.) gen (percent) cent) nitrogen V protease B I .ABLE 14.-EFFECT OF INCREASE OF ORGANIC NITROGEN IN COMBINATION OF OR- GANIC NITROGEN AND INORGANIC NITROGEN IN A SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY THE CULTIVATION OF ASPERGILLUS NIGER VAR. MACROSPOR US DBD-0406 I Composition of medium Ammonium Organic nitrosuliate (mg.) gen (percent) cen Inorganic nitrogen (per- Organic nitro- Productivity gen/inorganic of acid nitrogen protease B According to our studies on the production of acid )roteasc B of the known fungi by applying this new lOlId culture method, the strains belonging to Aspergilus niger group Aspergillus niger NRRL 334, Aspergillus ziger mut. shiemanni, Aspergillus carbonarius and the ike have been observed to show excellent results as hown in Tables 15 and 16.

lABLE 15.EFFECT OF INCREASE OF ORGANIC NITRO- GEN SOURCE IN A SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY THE CULTIVA- TION OF VARIOUS STRAINS BELONGING TO ASPER- GILLUS NIGER GROUP Productivity of acid protease B Bran: Defggtted soy- )omposition of medium Bren only can meal )rganic nitrogen (percent) 2. 2 5 tsp. niger var. macrosporus- 100 124 lsp. niger 100 182 lsp. niger mut. shiemanni- 100 217 lap. carbonarius 100 249 TABLE 16.-EFFECT OF INCREASE OF ORGANIC NITRO- GEN SOURCE IN A SOLID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY THE CULTI- VATION OF VARIOUS STRAINS BELONGING TO ASPER- Further we have succeeded to increase the production of acid protease B more than 40-60% or to make twice usual production in the liquid cultivation as shown in Tables 17-20 by making the organic nitrogen content in the medium containing organic nitrogen source only more than 0.4% by adding a considerable amount of organic nitrogen source or making organic nitrogen/inorganic nitrogen in the medium containing both organic and inorganic nitrogen sources above 6 by combined use of inorganic nitrogen source and organic nitrogen source, the inorganic nitrogen source such as ammonium sulfate being made below 0.06% of nitrogen content and the organic nitrogen source such as defatted soybean meal, defatted cotton seed meal, corn steep liquor being made above 0.4% by addition of a large amount.

TABLE 17.-EFFECT OF INCREASE OF ORGANIC NITROGEN SOURCE IN A LIQUID CUL- TURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY CULTIVATION OF ASPERGILLUS NIGER VAR. IMACROSPORUS i. Nitrogen source (percent)...

Organic Inorganic Organic nitro- Productivity Corn steep Deiatted soy- Ammonium nitrogen nitrogen gen/inorganic of acid liquor bean meal sulfate (percent) (percent) nitrogen protease B H TABLE 18.EFFECT OF INCREASE OF ORGANIC NITROGEN SOURCE IN A LIQUID CUL- TURE MEDIUM ON THE PRODUCTION-OF ACID PROTEASE B BY CULTIVATION OF ASPERGILLUS NIGER VAR.MACROSPORUS Organic Inorganic Organic nitro- Productivity Corn steep Defatted soy- Ammonium nitrogen nitrogen gen/inorganic of acid liquor bean meal sulfate (percent) (percent) nitrogen protease B 1 1 0. 1 0 100 1 5 0. 37 0 157 1 6 0. 44 0 183 1, 1,, Anti. 1.. -11 06 1 8 0. 54 0. 06 8. 6 151 TABLE 19.EFFECT OF INCREASE OF ORGANIC NITROGEN SOURCE IN A LIQUID CUL- TURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY CULTIVATION OF ASPERGILLUS NIGER VAR. MACROSPORUS Nitrogen source (percent) Organic Inorganic Organic nitro- Productivity Corn steep Defatted soy- Ammonium nitrogen nitrogen genin/organic of acid liquor bean meal sulfa (percent) (percent) nitrogen protease B TABLE 20.-EFFECT OF INCREASE OF ORGANIC NITROGEN SOURCE IN A LIQUID CUL- TURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY CULTIVATION OF ASPER GILL US NIGER VA R. MA CR OSPOR US Nitrogen source (percent) Organic Inorganic Organic nitro- Productivity Corn steep Defatted soy- Ammonium nitrogen nitrogen gen/inorganic of acid liquor bean meal sulfate (percent) (percent) nitrogen protease B In this case the combined use of defatted soy bean meal and corn steep liquor as the organic nitrogen source was observed to be more excellent than single use of them.

According to our studies on the production of acid protease B of the known fungi by applying this new liquid culture method, the strains belonging to Aspergillus niger group Aspergillus niger, Aspergillus niger mut, shieman'ni NRRL 361 and the like have been observed to show excellent results as shown in Table 21.

TABLE 21.-EFFECT OF INCREASE OF ORGANIC NITRO- GEN SOURCE IN A LIQUID CULTURE MEDIUM ON THE PRODUCTION OF ACID PROTEASE B BY THE CULTIVA- TION OF VARIOUS STRAINS BELONGING TO ASPER- GILLUS NIGER GROUP Productivity of acid protease B Corn steep liquor, Corn steep liquor,

l%+deiatted soy 1%+deiatted soy Nitrogen source bean meal, 3% bean meal 6% Organic nitrogen (percent) 0. 26 0. 49 Asp. niger var. macrosporus. 100 137 Asp. nz'ger 100 145 Asp. m'ger mut. shiemanni 100 207 The difference between acid protease A and B will be shown as follows:

ACTIVITY OF ACID PROTEASE A AND B EFFECT OFVARIOUS INORGANIC COMPOUNDS ON ACID PROTEASE A AND B Activity (percent) Concentration Compounds (M) Acid protease A Acid protease B A COMPARISON BETWEEN ACID PROTEASE A AND B ON THEIR VARI- OUS PROTEOLYTIC ACTIVITIES Acid protease A Acid protease B Enzyme Enzyme content Enzyme content Enzyme Substrates Opt. (mg.) activity Opt. (mg.) activtiy Soybean casein 1 2. 0 0. 500 0. 205 2. 6 0. 050 0. 110 Milk casein 1 2.0 0.500 0.923 2. 6 0.050 0. 809 Acid-denatured hemoglobin I 2. 0 O. 500 0. 608 2. 6 0. 050 0. 629 Urea-denatured hemoglobin l 3. 0 0. 500 0. 542 4. 5 0. 050 0. 489 Egg albumin 2. 0 1. 250 0. 406 2. 6 0. 0. 201 Wheat gluten 2.0 1. 250 0.277 2. 6 0. 125 0.479 Blood fibrin 2 1. 5 0.500 4. 85 2. 6 0. 500 6.05

Gelatin 3 3. 4 2. 000 68. 00 3. 4 2. 000 58. 00 Steer collagen 4 2. 6 0. 100 9. 69 3. 0 0. 200 7. 49

Enzyme activities were indicated as follows:

1 AO.D., 660 mu 1 AO.D., 2730311 1 (100 51m) (Who e solution) 4 sp (Supernatant of 30,000 r.p.m., 30 min.)

EFFECT OF VARIOUS ORGANIC COMPOUNDS ON ACID PROTEASE A AND B A A t (percent). M.

Concentra Acid Acid Iompounds tion protease A protease B 5 ISCOIbiO acid 10 M 153 119 my I I-bromosuccim'mide 10 M 4. I a-Chloromereuribenzoic acid. M 95. 5(94. 5) 79. 7(98. 5) (I) AMINO ACID COMPOSITION OF ACID PROTEASE A AND B 10- M 94. 5(65. 5) (8) 37(71. 5) FROM ASPERGILLUS NIGER VAR. MACROSPORUS, AND 10- M 106. 5 94. 5 PEPSIN 1 10' M 97.-5 92 104. 5 102 0 No. of residues per 1000 100 92 residues of amino acids 59. 4 71. 8 1.3 32.4 Y Acid Acid I Ammo acid protease A protease B I Pepsirf 10 M 101(88) 108(93. 5) 10 M 102 152 Aspartic acid 109 117 128 0.05% 94.5 08 15 Threonine 118 r .87 s2 10 M 115(26 6) 115(56 4) (2) Serine 131 140- 128 10- M 111 72. 5 Giutamlc acid 112 5 82 10- M 106 114 Pl'oiine 21 39 10- M 100 103. 7- Glycine- 115 V ----103 10" M 99. 3 109 Alanine 78 10- M 105. 4 83 Cystme (M (9) (4) 10 M 108 97. 5 20 Va1i11e 9 83 0. 05% 109 108 Methionme- 5 5 15 indium lauxylsulfate 10 M 69.2(0) (3) 0(0) Iso1eueine 37 40 79 ioybean trypsin inhibitor 0. 05% 99. 99. 6 Leucine 32 61 82 Paurocholate 10 M 102. 8(91.6) (I! 100(102) Tyrosine 52 52 Jrea. IHM 107 109 Phenylalanine" 38 41 41 Tryptophane-- 26 16 17 1 Lysine 17 31 3 LFFECT OF VARIOUS ANTIBIOTICS ON ACID PROTEASE 25 m 7 10 3 A AND B Ariuineun 2 7 11 0 Activity (percent) N 3 (108) Conoentm Acid v Acid (wigs-O. Blumenfeld and. G. E. Perlmann, J. Gen. PhysioL, 42, 553 Intlbiotics tion (M) protease A protease B I I I -aminopeuici]lanic amen---" 10 99.3 102.3 30 Jhloramphenicol 10- 109 104 hamieidin I 10' 99. 2 82. 5 Ianamyein. 10" 103. 8 105 Ienicillin G. 10- 95.4 107 tteptomycin 10- 101 101. 5 letracyciine 10- 108.8 97

0% 13% 35 2 1 1? 94 Acrd protease A and and protease B may be purely }g; 18? 83 lsolated from the crude enzyme powder by the processes shown m the followmg flow sheet.

CRUDE ENZYME POWDER PRECIPITATED WITH AMMONIUM SULFATE (50% SATURATION) I DESALTED & BUFFER-CHANGED WITH SEPHADEX G-25 CHROMATOGRAPHED WITH HYDROXYLAFATITF BUFFER: pH 0.0 PHOSPHATE BUFFER 0 002M 0 1M+0.5M N e]. W....... ,...I1...--...

FRACTION (I) FRACTION (11)? V 1* h M Q DESALTED & BUFFER-CHANGED DESAL'IED & BUFFER-CHANGED .3 WITH SEPHADEX G-25 WITILSEPHADEX, 07-25... j

CROMAIOGRAPHED WITH CHROMATOGRAPHED WITH; DEAE-CELLULOSE SE-CELLULOSE BUFFER: pH 6.0 PHOSPHATE BUFFER: 003 MCILVAINE BUFFER BUFFER 0.01M--0.1M+ 7 pH 3.0-7.0 IL 0.5M NBC]. ll DESALTED a. BUFFER-CHANGED DESALTED & BUFFER-CHANGED WITH SEPHADEX G-75 WITH SEPHADEX G-75 f BUFFER: pH 3.5 MCILVAINE BUFFER: pH 3.5 MCILVAINE BUFFER m- BUFFER ,.=0.oa F003 FREEZE-DRIED FREEZE-DRIED ACID PROTEASE A ACID PROTEASE B 13 The present invention will be explained more in detail by way of examples as follows:

EXAMPLE 1 Potato-sucrose agar slant cultures of the following strains were prepared. A mixture of bran 25 g. and defatted soy bean meal 25 g. was added with 60 ml. of water and put in 1 1. volume Erlenmyer flask which was then sterilized. After cooling, each strains from the slant cultures were inoculated in the sterilized culture media and cultivated at 33 C. for 4 days. The cultures were extracted with 500 ml. of water and the extracts were added with ammonium sulfate up to 75% saturation. The precipitates thus formed were dried to obtain the enzymes as follows:

Weight of pure enzyme Weight of calculated crude v enzyme Acid Acid 1 harvested protease A protease B Name of strain (g.) (mg.) (mg.)

Asperqillus nicer var.

macmsporus DBD-0406..." 12 55 308 Asperaillue m'ger IAM 2532.- 10 50 191 Aspergillus nicer mut.

shiemamti IAM 2531...-.. 12. 5 43 266 Aspergillus oureus var.

acidus IAM 2281 9 77 281 Aspergillusuureus var.

brevis IAM 2091 7 35 44 Aspergillus batatae IAM 2098.. 7. 5 23 192 Penicillium' frequentans 082; 5 53 78 Penicillium thomii IAM 7006. 4 11 35 EXAMPLE 2 Potato-sucrose agar slant cultures of the following strains were prepared. A mixture'of bran 25 g. and defatted soy bean meal 25 g. was added with 40 ml. of 5% aqueous solution of ammonium sulfate and put in 1 1. volume Erlenmyer flask which was then sterilized. Aftercoioling, each strains from the slant cultures were inoculatedin the sterilized culture media and cultured at 33., Cffor 4 days. The cultures were extracted with 500 :rnl fof "water and the extracts'were added with ammoniurn sulfate up to 75% saturation. The precipitates thus formed were dried to give the enzymes as follows:

Potato-sucrose agar slant cultures of the following strains were prepared. A liquid culture media composed of starcllip1%, corn steep liquor 1%, defatted soy bean meal 6%, ammonium sulfate 0.3%, K HPO 0.1% and CaCO 1% were separately put in 100 ml. volume Erlenmyer flasks 15 ml. apiece and sterilized. After cooling, each strains from the slant cultures were inoculated in the sterilized culture media and shake-cultured at 32 C. for 6 days. To the culture filtrates ammonium sulfate was added up to 75% saturation. The precipitates thus formed were dried to give the enzymes as follows:

The following strains were inoculated in a liquid culture media composed of starch 1%, corn steep liquor 1%, defatted soy bean meal 6%, K HPO 0.1% and CaCO 1% and cultured as in Example 1. The culture filtrates were subjected to salting out with ammonium sulfate to give the enzymes as follows:

Weight of pure enzyme Weight of calculated crude enzyme Acid Acid harvested protease A protease B Name of strain (g.) (mg.) (mg.)

Aspergillus 'nz'ger var.

macrosporus DBD-0406-. 14. 5 59 281 Aspergz'llus m'qer IAM 2532 22 59 344 Aspergillus aureus var.

brem's IAM 2001 12 45 225 We claim:

1. A process for simultaneously producing a new high acid protease (A) which has an optimum active pH at pH 2.0, 30 C. and exhibits high activity even at pH 1.5, 55 C., in addition to a usual acid protease (B) which has an optimum active pH at pH 2.6, 30 C. and substantially no activity at pH 1.5, 55 C. which comprises cultivating Aspergillus niger var. macrosporus DBD-0406 (ATCC- 16513) in a culture medium containing for a liquid culture above 0.4 percent of organic nitrogen and an effective amount but less than 0.06 percent of inorganic nitrogen such that the ratio of organic nitrogen to inorganic nitrogen is at least 6 and for a solid culture above 2.5 percent of organic nitrogen, and an etfective amount but less than 0.6 percent of inorganic nitrogen such that the ratio of organic nitrogen to inorganic nitrogen is at least 4.

2. A process for the production of acid protease B by a solid culture method as claimed in claim 1 in which an organic nitrogen source is used, the organic nitrogen content in the medium being more than 4% 3. A process as claimed in claim 2 in which an inorganic nitrogen source is also present, such that the ratio of organic nitrogen to inorganic nitrogen is at least 6.

4. A process for the produtcion of acid protease B by a liquid culture medium as claimed in claim 1 in which an organic nitrogen source is used, said source being selected from the group consisting of defatted soy bean meal and defatted cotton seed meal in combination with corn steep liquor.

References Cited UNITED STATES PATENTS 3,149,051 9/1964 Yoshida et al. --65 3,186,921 6/1965 Rupe 19'5-66 FOREIGN PATENTS 386,579 5/1963 Japan.

OTHER REFERENCES Koaze et al.: Agricultural and Biological Chemistry, vol. 28, No. 4, pp- 216-223, 1964.

LIONEL M. SHAPIRO, Primary Examiner US. Cl. X.R. 195-62 

